ABSTRACT
BACKGROUND: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. METHODS: The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. RESULTS: In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. CONCLUSIONS: NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.
Subject(s)
Pyrophosphatases , Triple Negative Breast Neoplasms , Animals , Female , Humans , Cell Line, Tumor , Cell Proliferation , Pyrophosphatases/genetics , Receptors, Estrogen/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathologyABSTRACT
Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.
Subject(s)
Protein Binding , Ligands , ProteolysisABSTRACT
Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.
Subject(s)
Anti-Infective Agents , Pyrimethamine , STAT3 Transcription Factor , Tetrahydrofolate Dehydrogenase , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Cell Line, Tumor , Folic Acid/metabolism , Humans , Proteome/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
STAT3 protein is a sought-after drug target as it plays a key role in the progression of cancer. Many STAT3 inhibitors (STAT3i) have been reported, but accumulating evidence suggests many of these act as off-target/indirect inhibitors of STAT signaling. Herein, we describe the STAT protein thermal shift assay (PTSA) as a novel target engagement tool, which we used to test the binding of known STAT3i to STAT3 and STAT1. This revealed STATTIC, BP-1-102, and Cpd188 destabilized both STATs and produced unique migratory patterns on SDS-PAGE gels, suggesting covalent protein modifications. Mass spectrometry experiments confirmed that these compounds are nonspecifically alkylating STATs, as well as an unrelated protein, NUDT5. These experiments have highlighted the benefits of PTSA to investigate interactions with STAT proteins and have helped reveal the novel reactivity of Cpd188. The described PTSA represents a promising chemical biology tool that could be applied to an array of other protein targets.
Subject(s)
STAT3 Transcription Factor , Signal Transduction , PhosphorylationABSTRACT
The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
Subject(s)
Pyrophosphatases/antagonists & inhibitors , Pyrophosphatases/metabolism , Binding Sites , Cell Line , Drug Design , Drug Development , Escherichia coli , Humans , Inorganic Pyrophosphatase/antagonists & inhibitors , Inorganic Pyrophosphatase/genetics , Inorganic Pyrophosphatase/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , Structure-Activity RelationshipABSTRACT
Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl-l-methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.
Subject(s)
Arginine/chemistry , Calorimetry, Differential Scanning/methods , Coenzymes/chemistry , Enzyme Assays/methods , Fluorometry/methods , Protein-Arginine N-Methyltransferases/chemistry , S-Adenosylmethionine/chemistry , Arginine/metabolism , Catalysis , Dimerization , Fluorescent Dyes/chemistry , Gene Expression , Kinetics , Methylation , Protein Conformation, alpha-Helical , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/isolation & purification , Protein-Arginine N-Methyltransferases/metabolism , Recombinant Proteins , S-Adenosylmethionine/metabolism , Substrate Specificity , TemperatureABSTRACT
Finding a cure for Alzheimer's disease is an urgent goal. Multifunctional metal binders are used to elucidate its pathological features and investigated as potential therapeutics. The use of physicochemical and TD-DFT calculations constituted successful strategy in the design of 1-(4-(benzo[d]oxazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL21) and 1-(4-(benzo[d]thiazol-2-yl)phenyl)-3-hydroxy-2-methylpyridin-4(1H)-one (HL22). We report the synthesis and full characterization of these compounds, including X-ray crystallography. Using fluorescent signal as the readout, it was determined that HL22 interacts with amyloid-beta protein fibrils, and permeates into bEnd.3 cells used as a mimic of the blood-brain barrier. This provides the first example of direct investigation of our hydroxypyridinone compounds within a biological setting.
Subject(s)
Amyloid beta-Peptides/metabolism , Pyridones/chemistry , Animals , Blood-Brain Barrier/drug effects , Cell Line/drug effects , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Fluorescence , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Structure , Permeability , Pyridones/pharmacologyABSTRACT
Computer-aided lead optimization derives a unique, orally bioavailable inhibitor of the signal transducer and activator of transcription (Stat)3 Src homology 2 domain. BP-1-102 binds Stat3 with an affinity (K(D)) of 504 nM, blocks Stat3-phospho-tyrosine (pTyr) peptide interactions and Stat3 activation at 4-6.8 µM, and selectively inhibits growth, survival, migration, and invasion of Stat3-dependent tumor cells. BP-1-102-mediated inhibition of aberrantly active Stat3 in tumor cells suppresses the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, VEGF, and Krüppel-like factor 8, which is identified as a Stat3 target gene that promotes Stat3-mediated breast tumor cell migration and invasion. Treatment of breast cancer cells with BP-1-102 further blocks Stat3-NF-κB cross-talk, the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule 1, macrophage migration-inhibitory factor/glycosylation-inhibiting factor, interleukin 1 receptor antagonist, and serine protease inhibitor protein 1, and the phosphorylation of focal adhesion kinase and paxillin, while enhancing E-cadherin expression. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of human breast and lung tumor xenografts.
Subject(s)
Breast Neoplasms/therapy , Lung Neoplasms/therapy , STAT3 Transcription Factor/pharmacokinetics , Administration, Oral , Animals , Biological Availability , Cell Line , Cell Line, Tumor , Female , Humans , Mice , STAT3 Transcription Factor/administration & dosage , Xenograft Model Antitumor AssaysABSTRACT
INTRODUCTION: The Trk family proteins are membrane-bound kinases predominantly expressed in neuronal tissues. Activated by neurotrophins, they regulate critical cellular processes through downstream signaling pathways. Dysregulation of Trk signaling can drive a range of diseases, making the design and study of Trk inhibitors a vital area of research. This review explores recent advances in the development of type II and III Trk inhibitors, with implications for various therapeutic applications. AREAS COVERED: Patents covering type II and III inhibitors targeting the Trk family are discussed as a complement of the previous review, Type I inhibitors of tropomyosin receptor kinase (Trk): a 2020-2022 patent update. Relevant patents were identified using the Web of Science database, Google, and Google Patents. EXPERT OPINION: While type II and III Trk inhibitor development has advanced more gradually compared to their type I counterparts, they hold significant promise in overcoming resistance mutations and achieving enhanced subtype selectivity - a critical factor in reducing adverse effects associated with pan-Trk inhibition. Recent interdisciplinary endeavors have marked substantial progress in the design of subtype selective Trk inhibitors, with impressive success heralded by the type III inhibitors. Notably, the emergence of mutant-selective Trk inhibitors introduces an intriguing dimension to the field, offering precise treatment possibilities.
Subject(s)
Drug Design , Drug Development , Patents as Topic , Protein Kinase Inhibitors , Signal Transduction , Humans , Animals , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Receptor, trkA/antagonists & inhibitors , Receptor, trkA/metabolism , MutationABSTRACT
Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.
Subject(s)
Antineoplastic Agents , Folic Acid Antagonists , Neoplasms , Humans , Pyrimethamine/pharmacology , Folic Acid Antagonists/pharmacology , Folic Acid Antagonists/chemistry , Methotrexate/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Biology , Tetrahydrofolate Dehydrogenase/chemistryABSTRACT
Thymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein-protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.
Subject(s)
Cytokines , Receptors, Cytokine , Thymic Stromal Lymphopoietin , Humans , Cytokines/metabolism , Receptors, Cytokine/metabolism , Receptors, Cytokine/antagonists & inhibitors , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/metabolism , Th2 Cells/immunology , Th2 Cells/drug effects , Th2 Cells/metabolism , Animals , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Protein Binding/drug effects , Interleukin-4/metabolism , Skin/drug effects , Skin/metabolism , Skin/pathology , Lung/metabolism , Lung/drug effects , Lung/pathology , Molecular Docking SimulationABSTRACT
INTRODUCTION: Trk inhibitors are significant in the realm of personalized medicine as they target specific genetic alterations, such as NTRK gene fusions, leading to improved treatment outcomes for cancer patients. By tailoring the treatment to the genetic characteristics of the tumor rather than the tumor type, Trk inhibitors offer the potential for more effective and precise therapies, resulting in enhanced response rates and prolonged survival for patients with NTRK fusion-positive cancers. AREAS COVERED: Patents covering type I inhibitors targeting the Trk family are discussed, building upon our prior review series on Trk inhibitors. Relevant patents were identified through the Web of Science database, Google, and Google Patents. EXPERT OPINION: The field of Trk inhibitors has evolved significantly, as reflected in the current patent literature, which emphasizes the selective structural refinement of clinical champions. Efforts now concentrate on enhancing efficacy against on-target resistance mechanisms, with modifications made to improve potency, reduce toxicity, and enhance pharmacokinetics. Combination therapies show potential to address off-target resistance mechanisms and improve treatment outcomes. Challenges remain in accurately diagnosing NTRK gene alterations and integrating screening into routine clinical practice. Trk inhibitors have surpassed their conventional role of inhibition and are now seeing new applications in radiopharmaceutical development and as molecular targeting agents.
Subject(s)
Neoplasms , Receptor, trkA , Humans , Tropomyosin/therapeutic use , Protein Kinase Inhibitors/pharmacology , Patents as Topic , Neoplasms/drug therapyABSTRACT
Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.
ABSTRACT
A small molecule drug with poor aqueous solubility can be conjugated to a hydrophilic polymer like poly(ethylene glycol) (PEG) to form an amphiphilic polymer-drug conjugate that self-assembles to form nanoparticles (NPs) with improved solubility and enhanced efficacy. This strategy has been extensively applied to improve the delivery of several small molecule drugs. However, very few reports have succeeded to tune the rate of drug release from these NPs. To the best of our knowledge, there have been no reports of utilizing click and steric hindrance chemistry to modulate the drug release of self-assembling polymer-drug conjugates. In this study, we utilized click chemistry to conjugate methoxy-PEG (mPEG) to an anti-tumor drug, paclitaxel (PTX). A focused library of PTX-Rx-mPEG (x = 0, 1, 2) conjugates were synthesized with different chemical modalities next to the cleavable ester bond to study the effect of increasing steric hindrance on the self-assembly process and the physicochemical properties of the resulting PTX-NPs. PTX-R0-mPEG had no added steric hindrance (x = 0; minimal), PTX-R1-mPEG consisted of two methyl groups (x = 1: moderate), and PTX-R2-mPEG consisted of a phenyl group (x = 2: significant). Drug release studies showed that PTX-NPs released PTX at a decreased rate with increasing steric hindrance. Pharmacokinetic studies showed that the AUC of released PTX from the moderate-release PTX-R1-NP was approximately 20-, 6-, and 3-fold higher than that from free PTX, PTX-R0-NP and PTX-R2-NP, respectively. As a result, among these different PTX formulations, PTX-R1-NP showed superior efficacy in inducing tumor regression and prolonging the animal survival. The tumors treated with PTX-R1-NP displayed the lowest tumor progression markers (Ki68 and CD31) and the highest apoptotic marker (TUNEL) compared to the others. This work emphasizes the importance of taking a systematic approach in designing self-assembling polymer drug conjugates and highlights the potential of utilizing steric hindrance as a tool to tune the drug release rate from such systems.
Subject(s)
Antineoplastic Agents , Nanoparticles , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival , Drug Carriers/chemistry , Drug Delivery Systems , Drug Liberation , Esters , Nanoparticles/chemistry , Paclitaxel/therapeutic use , Polyethylene Glycols/chemistry , Polymers/chemistryABSTRACT
Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that is aberrantly activated in numerous human cancers. Inhibitors of activated Stat3-Stat3 protein complexes have been shown to hold therapeutic promise for the treatment of human cancers harboring activated Stat3. Herein, we report the design and synthesis of a focused library of salicylic acid containing Stat3 SH2 domain binders. The most potent inhibitor, 17o, effectively disrupted Stat3-phosphopeptide complexes (K(i)=13 µM), inhibited Stat3-Stat3 protein interactions (IC(50)=19 µM) and silenced intracellular Stat3 phosphorylation and Stat3-target gene expression profiles. Inhibition of Stat3 function in both breast and multiple myeloma (MM) tumor cells correlated with induced cell death (EC(50)=10 and 16 µM, respectively).
Subject(s)
Antineoplastic Agents/pharmacology , Cell Membrane Permeability/drug effects , STAT3 Transcription Factor/antagonists & inhibitors , Salicylic Acid/pharmacology , src Homology Domains/drug effects , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Design , Drug Screening Assays, Antitumor , Humans , Ligands , Models, Molecular , Molecular Structure , Molecular Weight , STAT3 Transcription Factor/metabolism , Salicylic Acid/chemical synthesis , Salicylic Acid/chemistry , Small Molecule Libraries , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Atopic diseases refer to common allergic inflammatory diseases such as atopic dermatitis (AD), allergic rhinitis (AR), and allergic asthma (AA). AD often develops in early childhood and may herald the onset of other allergic disorders such as food allergy (FA), AR, and AA. This progression of the disease is also known as the atopic march, and it goes hand in hand with a significantly impaired quality of life as well as a significant economic burden. Atopic diseases usually are considered as T helper type 2 (Th2) cell-mediated inflammatory diseases. Thymic stromal lymphopoietin (TSLP), an epithelium-derived pro-inflammatory cytokine, activates distinct immune and non-immune cells. It has been shown to be a master regulator of type 2 immune responses and atopic diseases. In experimental settings, the inhibition or knockout of TSLP signaling has shown great therapeutic potential. This, in conjunction with the increasing knowledge about the central role of TSLP in the pathogenesis of atopic diseases, has sparked an interest in TSLP as a druggable target. In this review, we will discuss the autocrine and paracrine effects of TSLP, how it regulates the tissue microenvironment and drives atopic diseases, which provide the rationale for the increasing interest in TSLP as a druggable target.
Subject(s)
Cytokines/drug effects , Cytokines/metabolism , Hypersensitivity/physiopathology , Age Factors , Biological Products/pharmacology , Cellular Microenvironment/physiology , Cytokines/genetics , Disease Progression , Humans , Inflammation Mediators/physiology , Janus Kinases/metabolism , Protein Isoforms , Severity of Illness Index , Signal Transduction/physiology , Th2 Cells/metabolism , Thymic Stromal LymphopoietinABSTRACT
The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.
ABSTRACT
Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Ganciclovir/pharmacology , Pyrophosphatases/metabolism , Antiviral Agents/chemistry , Cell Line, Tumor , Female , Ganciclovir/chemistry , Humans , Hydrolysis , Microbial Sensitivity Tests , Recombinant Proteins/metabolismABSTRACT
The transcription factor STAT3 plays a key role in cancer and immunity, being widely explored as a potential drug target for the development of novel immunomodulatory or anticancer therapeutics. The mechanisms of small molecule-derived inhibition of STAT3 appear, however, to be more complex than initially perceived. Our recent discovery, that some novel STAT3 inhibitors were bona fide inhibitors of the cytosolic selenoprotein oxidoreductase TrxR1 (TXNRD1), led us to explore the effects of a wide array of previously described STAT3 inhibitors on TrxR1 function. We found that 17 out of 23 tested STAT3 small molecule inhibitors indeed inhibited purified TrxR1 at the reported concentrations yielding STAT3 inhibition. All tested compounds were electrophilic as shown by direct reactivities with GSH, and several were found to also be redox cycling substrates of TrxR1. Ten compounds previously shown to inhibit STAT3 were here found to irreversibly inhibit cellular TrxR1 activity (Auranofin, Stattic, 5,15-DPP, Galiellalactone, LLL12, Napabucasin, BP1-102, STA-21, S3I-201 and Degrasyn (WP1130)). Our findings suggest that targeting of TrxR1 may be a common feature for many small molecules that inhibit cellular STAT3 function. It is possible that prevention of STAT3 activation in cells by several small molecules classified as STAT3 inhibitors can be a downstream event following TrxR1 inhibition. Therefore, the relationship between TrxR1 and STAT3 should be considered when studying inhibition of either of these promising drug targets.